The polymerase chain reaction (PCR) has become the conventional technique used to amplify specific DNA or RNA sequences. U.S. Pat. No. 4,683,202, issued Jul. 28, 1987 to Mullis and U.S. Pat. No. 4,683,195, issued Jul. 28, 1987 to Mullis et al. describe the basic PCR technique. Since the first disclosure of the PCR method, it has had a profound effect on the practice of biotechnology and biomedical science. More than a thousand subsequently-issued U.S. patents reference one or both of these disclosures.
Typically, the amplification of a DNA sequence is performed by first selecting and obtaining two oligonucleotide primers complementary ends of a target DNA sequence. The primers, a polymerase enzyme, a mixture of the four common nucleotide triphosphates, various salts and buffers are mixed with the target DNA which is heated above about 90° C. to denature the DNA, separating the target double-stranded DNA into single-stranded DNA templates. Annealing (i.e. sequence-specific hybridization or binding) of the primers to the ends of the DNA templates is promoted by slowly cooling the reaction mixture to less than about 60° C. The temperature is then raised above about 70° C. for a period of replication, a process also known as primer extension. The polymerase reads each DNA template strand in the 3′ to 5′ direction, synthesizing a complementary strand from the ends of the primers in the 5′ to 3′ direction. This completes one cycle of DNA amplification, which creates starting material for a new cycle. With each complete cycle of denaturation, primer annealing, and primer extension, the process generates an exponentially increasing (2n) number of copies of the original, target DNA sequence. To begin a new cycle, the reaction mixture is again heated above 90° C. to denature the double-stranded product into single-stranded DNA templates. The primer annealing and extension steps are then repeated.
This basic PCR amplification scheme, together with various extensions and modifications, enables many different methods for the manipulation and detection of nucleic acids, including for example diagnostic and forensic assays, which require the creation of a threshold amount of DNA from a small initial sample. PCR technology is used, for example, in infectious and genetic disease monitoring, DNA and RNA sequencing, gene expression studies, drug development, and forensic fingerprinting. This has become the standard technology for the detection, identification, and quantification of viral and bacterial pathogens. Several PCR-based diagnostic tests are available for detecting and/or quantifying pathogens, for example, including: HIV-1, which causes AIDS; hepatitis B and C viruses, which can cause liver cancer; human papillomarvirus, which can cause cervical cancer; RSV, which is the leading cause of pneumonia and bronchiolitis in infants; Chlamydia trachomatis and Neisseria gonorrhoeae, which can lead to pelvic inflammatory disease and infertility in women; cytomegalovirus, which can cause life-threatening disease in transplant patients and other immuno-compromised people; and, Mycobacterium tuberculosis, which causes cough and fatigue in its active state and can irreversibly damage infected organs. However, despite addressing needs in numerous areas, current PCR and PCR-based technologies still suffer from several substantial limitations.
Limitations of Conventional PCR and PCR-Based Technologies
Fidelity: Accuracy on normal sequences limits conventional PCR. For example, Taq, a thermostable polymerase commonly used for DNA amplification, exhibits an error rate of approximately 1×10−4 errors/base pair during PCR. This means that the PCR amplification of a 400 base pair DNA sequence will randomly introduce approximately 40,000 errors among all molecules in the PCR product over 20 cycles.
Accuracy on difficult target sequences (e.g. GC rich or repeat sequences) is an even more significant limitation of conventional PCR and PCR-based technologies. The error rate for conventional polymerase enzymes such as Taq, depends strongly on the target nucleotide sequence. For example, when the sequence is G+C rich (as seen for example in the 5′ regulatory region of the chicken avidin gene), PCR with Taq is oftentimes not a viable process. Likewise, simple repeating sequences, such as trinucleotide repeats (AGC)n or other tandem repeats (A)n, can increase Taq's error rate to 1.5×10−2 errors per repeat sequence. See, Shinde et al., Nucleic Acids Research, 31:974. For this reason, several patents have been issued for polymerases that have been genetically engineered to have incrementally higher fidelity (i.e. lower error rates). These include Hi-Fidelity and Phusion Polymerases.
Length Limitations: The length of the target sequence to be amplified also limits current PCR techniques. Although a few reports have claimed amplification of sequences up to 10 to 20 kilobases, this is highly unusual and quite difficult in routine practice. Moreover, PCR amplification of long target templates is only possible on a limited set of well-behaved DNA sequences. The practical upper-limit for fairly routine and cost-effective amplification of DNA on well-behaved sequences is about 300 to 400 bases in length and is generally reduced for sequences having high G-C content.
Limited Amplification: Current PCR techniques are also limited in the number of amplification cycles that can be carried out in a reaction mixture. Repeated heating and cooling cycles result in progressive enzyme degradation, which limits the factor by which starting material can be amplified. Conventional PCR amplification can rarely be extended beyond 30-35 cycles.
Robustness: Conventional PCR typically requires significant volumes of reagents, bulky equipment (e.g., thermal cyclers), substantial human labor (e.g., tedious optimizations), and minimum amounts of starting material, each of which contributes to making conventional PCR a costly and time-consuming process. Current PCR techniques typically take from several hours for normal sequences to several days to weeks for difficult sequences or long template. Conventional PCR requires a significant amount of time to cycle and equilibrate the temperature of the reaction mix. Moreover, time-consuming optimizations can be required in order to reliably amplify targets that are less than ideal.
Tightly controlled conditions (e.g., temperature, pH, and buffer ingredients) are required for performance of conventional PCR techniques. Additionally, various contaminants can interfere with PCR amplification by directly inhibiting or interfering with polymerase enzymes used to copy the target DNA or RNA. This further limits the quality of starting material that can be used for amplification and places additional requirements on the level of purity that must be obtained by DNA or RNA extraction techniques before the amplification steps can be reliably performed. The performance environment of conventional PCR is generally limited to laboratories, and is rarely practicable in remote locations, doctor's offices, at the patient's bedside, or out in the field.
Sensitivity and Specificity of Diagnostics: The sensitivity of PCR-based diagnostic and forensic kits and assays depends on the overall yield, accuracy, robustness, and target length achievable in a PCR reaction. The above-mentioned limitations in performance parameters of current PCR set limits on the minimum amount of starting DNA or RNA necessary in order to reliably carry out PCR amplification. This, in turn, limits the sensitivity of any pathogen detection system, diagnostic, or forensic kits or assays that rely upon conventional PCR or PCR-based technologies. The specificity of a PCR-based diagnostic, forensic, or pathogen detection system depends critically on the accuracy with which DNA can be amplified and read as well as the length of the target DNA or RNA that can be reliably amplified and identified.
For these and other reasons, current generation PCR-based technologies and detection systems are generally limited with respect to overall speed, efficiency, cost-effectiveness, and scope of use. Incremental improvements to conventional PCR methods and devices have been proposed with respect to some of the isolated performance parameters described above. For example, Tso et al. discloses a PCR microreactor for amplifying DNA using microquantities of sample fluid in U.S. Pat. No. 6,613,560, issued Sep. 2, 2003. Alternatives to high temperature DNA denaturation have also been proposed. For example, Purvis disclosed a method of electrochemical denaturation of double-stranded nucleic acid in U.S. Pat. No. 6,291,185, issued Sep. 18, 2001. Stanley discloses another method of electrochemical denaturation of nucleic acids in U.S. Pat. No. 6,197,508, issued Mar. 6, 2001. Dattagupta et al. have disclosed a method of using primers to displace the DNA strand from the template in U.S. Pat. No. 6,214,587, issued Apr. 10, 2001. Mullis, supra, suggested the use of helicase enzymes for separating DNA strands.
In view of the limitations of conventional PCR, and despite the proposal of various incremental improvements, there remains a need in the art for improved methods, devices, and compositions for the amplification, manipulation, sequencing, and detection of nucleic acids.